游仆虫Rab蛋白基因的克隆表达与纯化

为对单细胞原生动物纤毛虫中Rab蛋白的功能进行研究 ,进而探讨以胞吞和胞吐为主要物质交换途径的纤毛虫中囊泡定向运输的机理 .利用PCR技术从游仆虫大核DNA及cDNA中扩增出rab基因 ,并进行了序列分析 ,该基因全长为 783bp ,两端为端粒序列 ,编码框为 6 2 4bp ,编码 2 0 7个氨基酸 ,开放读框中有 3个TGA ,在此编码半胱氨酸 .利用定点突变将rab基因中 3个TGA突变为通用半胱氨酸密码子TGC .将游仆虫Rab蛋白基因构建于原核表达载体pGEX 4T 2中 ,得到的重组质粒pGEX Eorab1转化至大肠杆菌BL2 1(DE3)中 ,IPTG诱导表达 .表达产物与抗GST抗体在 4 9kD处有很强的交叉反应 .融合蛋白GST EoRab1通过亲和层析柱纯化和凝血酶的切割 ,再经两步纯化得到电泳纯的游仆虫Rab蛋白 .

Cloning,Expression and Purification of a Euplotes rab Gene in E.coli

To study the function of Rab protein of unicellular protozoa ciliates and to understand further the mechanism of the vesicle traffic in ciliates, which transports material mainly by endocytosis and exocytosis. The rab gene was amplified from macronuclear DNA and cDNA of Euplotes octocarinatus. The gene was 783 bp with an ORF of 624 bp encoding EoRab1 protein and containing three in frame TGA codes. Three TGA were successfully mutated to TGC by site direct PCR procedure. Recombinant expression plasmid pGEX Eorab1 was constructed. GST EoRab1 fusion proteins were highly expressed in E. coli BL21(DE3) by IPTG induction. Western blotting with anti GST antibodies assay confirmed that putative fusion protein with molecular mass of 49 KD was the expected GST EoRab1. GST EoRab1 protein was purified by GST affinity chromatography. EoRab1 protein was obtained by cleaving GST EoRab1 protein with thrombin protease and the purity of EoRab1 protein reached 95 percent.